Single cell regulatory architecture of human pancreatic islets suggests sex differences in β cell function and the pathogenesis of type 2 diabetes

Type 2 and type 1 diabetes (T2D, T1D) exhibit sex differences in insulin secretion, the mechanisms of which are unknown. We examined sex differences in human pancreatic islets from 52 donors with and without T2D combining single cell RNA-seq (scRNA-seq), single nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq), hormone secretion, and bioenergetics. In nondiabetic (ND) donors, sex differences in islet cells gene accessibility and expression predominantly involved sex chromosomes. Islets from T2D donors exhibited similar sex differences in sex chromosomes differentially expressed genes (DEGs), but also exhibited sex differences in autosomal genes. Comparing β cells from T2D vs. ND donors, gene enrichment of female β cells showed suppression in mitochondrial respiration, while male β cells exhibited suppressed insulin secretion. Thus, although sex differences in gene accessibility and expression of ND β cells predominantly affect sex chromosomes, the transition to T2D reveals sex differences in autosomes highlighting mitochondrial failure in females.


Introduction
Type 1 and type 2 diabetes (T1D, T2D) are heterogeneous diseases and biological sex affects their pathogenesis.In the context of T2D, sex affects the development of adiposity, insulin resistance, and dysfunction of insulin-producing β cells of pancreatic islets. 1,24][5] A missense mutation in the β cell-enriched MAFA transcription factor is found in subjects with adult-onset β cell dysfunction, where men are more prone to β cell failure than women. 6Similarly, T1D is the only common autoimmune disease characterized by a male predominance 1,7,8 , and males who develop T1D during puberty have lower residual β cell function than females at diagnosis. 9Furthermore, among T1D subjects receiving pancreatic islet transplantation, recipients of male islets exhibit early graft β cell failure when compared to recipients of female islets. 10The mechanisms that drive preferential β cell failure in males, however, is unknown.Studying sex differences in islet biology and dysfunction represent a unique avenue to understand sex-specific heterogeneity in β cell failure in diabetes. 22][13][14][15][16][17][18][19] However, the sex-specific and cell autonomous factors that influence islet function outside the in vivo hormonal environment are unknown.These differences could be due to sex chromosome gene dosage, or epigenetic programming caused by testicular testosterone during development in males. 1,20,21The Genotype-Tissue Expression (GTEx) project analysis of the human transcriptome across various tissues revealed that the strongest sex bias is observed for X-chromosome genes showing higher expression in females. 22In the pancreas, the majority of genes with sex-biased expression are on the sex chromosomes and most sex-biased autosomal genes are not under direct influence of sex hormones. 23In human pancreatic islets, DNA methylation of the X-chromosome is higher in female than males. 24Thus, the cell autonomous influence of sex chromosome genes may impact sex-specific islet biology and dysfunction and diabetes pathogenesis.
Here, we examined sex and race differences in human pancreatic islets from up to 52 donors with and without T2D using an orthogonal series of experiments including single cell RNA-seq (scRNA-seq), single nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq), and dynamic hormone secretion and bioenergetics.Our studies establish biological sex as a genetic modifier to consider when designing experiments of islet biology.

Human islet cells show conserved autosomal gene expression signatures independent of sex and race.
We performed scRNA-seq on pancreatic islets from age-and BMI-matched non-diabetic donors across race and sex (Tulane University Islet Dataset, TUID, n=15), which we combined with age-and BMI-matched non-diabetic donors and donors with T2D from the HPAP database 25,26 (n=37) to create an integrated atlas of islet cells (Fig. 1a and Extended Data Fig. 1a-b).To obtain high-quality single cell signatures, we used a series of thresholds including filtering, ambient RNA correction, and doublet removal, resulting in 141,739 high-quality single cell transcriptomes, with TUID showing optimal sequencing metrics (Extended Data Fig. 1c and d).We identified 17 cell clusters, which we annotated based on marker genes with differential expression (DEGs) correlating to known transcriptional signatures of islet cells (Fig. 1b). 27Cell clusters showed even distribution across sex, race, disease, and library of origin (Fig. 1c).Consistent with a prior analysis 25 , all islet cell clusters except for lymphocytes and Schwann cells were identified in HPAP data (Extended Data Fig. 1b).Notably, we observed greater variability in total cell number within each donor library in HPAP compared to TUID (Fig. 1d).We observed a high degree of correlation between cell-specific gene expression and cell clusters across donors (Extended Data Fig. 1e).As expected, sex chromosome-specific transcripts were expressed across male and female cell types (Extended Data Fig. 1f).
We more broadly examined DEGs across clusters by creating sample 'pseudo-bulk' profiles for each cell type to control for pseudo-replication of cells being repetitively sampled from a fixed donor.For example, each β cell per donor was aggregated into one profile, enabling us to control for the disproportionate β cell numbers across donors (Fig. 1d).Autosomal genes with expression specific to each cell cluster were consistent across sex and race.In endocrine cell types, we found 5,481 β (INS, MAFA), 7,395 α (GCG, ARX), 71 δ (HHEX, SST), 3 ε (GHRL), 12 γ (PPY) and 159 cycling endocrine (TOP2A, MKI67) DEGs (Fig. 1e-f and Extended Data Fig. 1g).

Sex differences in islet cell transcriptomes from non-diabetic donors predominantly affect sex chromosome genes.
We performed two sets of analysis comparing changes in gene expression in biological variables of sex and race across groups.To study transcriptional differences across donors, we generated principal component analysis (PCA) plots of islet 'pseudo-bulk' transcriptional profiles across all 52 donors.Donors did not cluster based on sex, race, disease status, or origin of donor (Fig. 2a).We next segregated donors by cell type, and the resulting PCA showed clustering of samples based on cell type (Fig. 2b).Both whole islet 'pseudo-bulk' and individual cell type 'pseudo-bulk' sample profiles showed no clustering based on sex or race.This suggests that human islets likely do not have major differences in cell type transcriptional profiles across either race or sex.
Focusing on non-diabetic donors, we examined genes with differences in expression between sexes using cell type 'pseudo-bulk' analysis.Most sex-associated genes were related to sex chromosomes (FDR<0.1).In β cells, 60% of genes with increased expression in females were linked to the X chromosome and 70% of genes increased in males were linked to the Y chromosome (Fig. 2c and Extended Data Fig. 2a).Similarly, in α cells 50% of male-and 57% of female-enriched genes were linked to the X or Y chromosome, respectively (Fig. 2d and Extended Data Fig. 2a).In α/β cells, X-inactive specific transcript (XIST) and lysine demethylase 6A (KDM6A) were upregulated in females, while ribosomal protein S4 Y-linked 1 (RPS4Y1) and lysine demethylase 5C (KDM5D) was upregulated in males (Fig. 2c and d).We only observed significant race differences in DEGs between hispanic and white β and α cells (Extended Data Fig. 2c).
Next, we identified sex-specific changes in pathways related to sex chromosome genes using gene set enrichment analyses (Fig. 2e and Extended Data Fig. 2b).Female β cells were enriched for pathways for Xchromosome inactivation and histone lysine demethylation, whereas male β cells were enriched for pathways for Y-chromosome genes, histone lysine demethylation, and male sex determination (Fig. 2e).Female α cells were enriched for histone lysine demethylation, X-chromosome inactivation, and mitochondrial transcription, while male α cells were enriched for histone demethylase activity (Fig. 2f).Similar effects were observed in other cell types (Extended Data Fig. 2b).Race differences in islet cells are shown in Fig. 2e and f as well as Extended Data Fig. 2c and d.Of note, black male β cells showed higher cytokine signaling compared to white males, suggesting black male β cells may exhibit a higher inflammatory response (Fig. 2e).

Accessible chromatin landscape across islet cells
To examine the effect of sex on the epigenome, we performed snATAC-seq on all non-diabetic donors of the TUID.To confirm library quality, we filtered and evaluated single nuclei across all 15 donors for TSS enrichment, fragment of reads in promoters, and fragment reads in accessible peaks (Extended Data Fig. 3a and b), as well as sample specific sequencing metrics (Extended Data Fig. 3c and d).We then clustered the 52,613 filtered profiles resulting in 11 distinct cell clusters which, like gene expression data, were evenly distributed across sex, race, and donor (Fig. 3a-c).To determine the identity of each cluster, we used label transfer to annotate each snATAC-seq cell cluster using our integrated scRNAseq islet cell atlas as a reference.We observed a high degree of correlation between genes with differential accessibility in snATAC-seq and genes with differential expression scRNAseq (Fig. 3d).Cell types also showed a high degree of correlation between RNA expression, chromatin accessibility, and predicted RNA expression (Extended Data Fig. 3e-g).We further examined the cell type annotations using the activity of cell type-specific genes.This validated clusters representing β (INS-IGF2), α (GCG), δ (SST), γ (PPY), acinar ductal (CFTR), (PRSS1), endothelial (ESM1), macrophage (SDS), stellate PDGFRA) and lymphocyte (CD3D) cells by comparing gene accessibility with predicted RNA expression (Fig. 3e and f, Extended Data Fig. 3h).
To characterize regulatory programs across each cluster, we identified candidate cis-regulatory elements (cCREs) in each cell type resulting in 404,697 total cCREs across all 11 cell types.We next identified cCREs with activity specific to each cell type, resulting in 55,710 cell type-specific cCREs (Fig. 3g).We identified genes in proximity to cell type-specific cCREs, resulting in a list of putative gene targets of cell type-specific regulatory programs.Evaluating these gene sets for enrichment of gene ontology terms revealed cell type-specific processes, and which were similar to those identified in cell type-specific gene expression (Fig. 3h).Using chromVAR 29 , we identified transcription factor (TF) motifs enriched in the accessible chromatin profiles of each cell type using the JASPAR 2020 database. 30In-depth analysis of these motifs revealed cell type-specific TF motif enrichment patterns (Fig. 3i).For example, we observed enriched motifs for ISL1 in endocrine cells, PDX1 in β and δ cells, and SOX9 in ductal and acinar cells (Fig. 3i and j).These accessible motifs also paralleled cell type specific TF expression in scRNA-seq (Fig. 3j).Similar to previous studies [31][32][33][34] , hierarchical motif clustering highlighted that the regulatory programs of β and δ cells are more related, as with α and γ cells (Fig. 3g).Select motifs highly enriched for a cell type (fold enrichment>1.5,-log10 FDR>50) included PAX4, RFX2, NKX6-2 and PDX1 in β cells, NKX6-2, NKX6-1, PDX1, and MEOX1 in δ cells, MAFB, FOXD2 and GATA2-5 in α cells, and KLF15 and NRF1 in γ cells (Extended Data Fig. 3i).Non-endocrine cells motif enrichments are also provided in Extended Data Fig. 3i.

Sex differences in chromatin accessibility of islet cells from non-diabetic donors predominantly affects sex chromosomes
To assess sex differences in chromatin accessibility, we identified sex-associated cCREs using logistic regression.As expected, β cells exhibited sex differences in chromatin accessibility at sex chromosome genes including KDM6A, XIST and KDM5D (Fig. 4a).Males exhibited more differentially accessible regions (250 in β, 565 in α) than females (203 in β, 553 in α).Next, we identified genes in a 100 kb proximity to sex-associated cCREs and interrogated their RNA expression.We found that Y-linked genes (SRY, RPS4Y1, UTY, TTTT14) in males and X-linked genes (KDM6A, XIST, DHRSX) in females were proximal to sex-associated cCREs (Fig. 4b).Accordingly, when comparing gene expression and cCREs with sex-specific association, we predominantly observed sex-chromosome genes (Fig. 4c).Gene ontology analysis of this subset of genes revealed enrichment in pathways regulating epigenetic control and X chromosome dosage compensation in females, and histone modification in males (Fig. 4d).Notably, the histone demethylase X-linked gene KDM6A and the long non-coding RNA XIST were more accessible in female islet cells, while the histone demethylase Y-linked gene KDM5D was more accessible in males (Fig. 4e).We examined sex differences in TF-specific motif accessibility in α/β cells.
Notably, females exhibited a greater number of TF-specific accessible motifs (511 in β, 376 in α) compared to males (33 in β, 74 in α) (Fig. 4f).Upon interrogating differentially expressed TF across cell types, MAFA, SIX3, PDX1, and RXRG were upregulated in β cells while ARX, FEV, STAT4 and ISL1 were upregulated in α cells irrespective of sex (Fig. 4g).We applied Pando 35 to scRNA-seq and snATAC-seq data to infer relationships between target gene expression, TF activation, and TF binding and define gene regulatory networks (GRNs) in male and female β and α cells.The GRNs provide sets of regulated target genes and cCREs for expressed TFs.

Sex and race differences in β cell function
We performed dynamic insulin and glucagon secretion assays in TUID islets for non-diabetic donors.We observed a decreased insulin response to high glucose and IBMX (a phosphodiesterase inhibitor which raises intracellular cAMP) in black male compared to white male islets (Fig. 5a and b).There was no significant difference in insulin secretion across sex and race using other classical insulin secretagogues (Fig. 5a-d) or an ascending glucose concentration ramp (Extended Data Fig. 4a-d).We observed no difference of race or sex on α cell function using classical glucagon secretagogues, although females exhibited a trend toward higher glucagon secretion (Fig. 5e-h).We also examined the effects of sex and race on islet bioenergetics by quantifying oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) during a glucose challenge in TUID islets.Female islets exhibited greater ATP-mediated respiration and coupling efficiency than male islets (Fig. 5i-l), suggesting more efficient mitochondria.There was no difference in ECAR between male and female islets (Extended Data Fig. 4e-h).
Dysregulation of β and α cell transcriptomes from non-diabetic compared with T2D donors suggests sex differences in T2D pathogenesis.
We examined the effect of sex on islet hormone secretion using the HPAP islet perifusion database matched for donors we sequenced in this study.Islets from male and female donors with T2D exhibited decreased insulin secretion in response to high glucose, incretin and KCl compared to islets from non-diabetic donors (Extended Data Fig. 5a and b), without evidence for sex difference.T2D islets exhibited no difference in α cell function in hypoglycemic conditions compared to non-diabetic donors (Extended Data Fig. 5c and d).
We compared the transcriptional profile of male and female HPAP donors with T2D.In contrast with non-diabetic donors, where most sex-associated genes were related to sex chromosomes (Fig. 2c and d), islets from T2D donors exhibited multiple sex-specific differences in DEGs from sex chromosomes and autosomes (Fig. 6a).
When comparing DEGs in β and α cells from male and female T2D donors, the largest and most significant changes were restricted to sex-linked genes (Fig. 6b).We next compared the transcriptional profile of male and female HPAP donors with T2D to that of non-diabetic TUID and HPAP donors (Extended Data Fig. 1a).Notably, in comparison of T2D vs. non-diabetic β cells, females exhibited more DEGs from autosomes (721 upregulated and 1164 downregulated) than males (111 upregulated and 99 downregulated), with only 5.2% of DEGs shared across sex (Fig. 6c and d).Similarly, in comparison of T2D vs. non-diabetic α cells, females exhibited more DEGs from autosomes (589 upregulated and 1552 downregulated) than males (14 upregulated and 6 downregulated), with only 0.28% overlap (Fig. 6c and f).When comparing T2D vs. non-diabetic donors in other cell types, females also exhibited more autosomal DEGs than males (Fig. 6c).We determined enrichment of gene ontology terms in these genes, and female β and α cells exhibited reduced mitochondrial function and respiration pathways in T2D (Fig. 6e and g) while male β cells exhibited reduced hormone and insulin secretion pathways in T2D (Fig. 6e).Enrichment of ontology terms for other islet cells in females and males are shown in Extended Data Fig. 6.

Sex-and cell-specific differences in T2D associated genetic risk
While sex-stratified genome-wide association studies (GWAS) have been performed for T2D, the specific celltypes contribution to disease risk at each disease-associated locus remain unknown.To address this, we performed genomic enrichment analyses of our snATAC-seq open chromatin regions in T2D, fasting glucose, and fasting insulin GWASs using LD score regression.All islet endocrine cell types showed significant genomic enrichment (FDR < 0.05) in both male and female T2D GWAS, suggesting common endocrine-driven mechanisms at disease risk loci (Figure 7a and Extended data Fig. 7a).Notably, macrophages, lymphocytes, and quiescent stellate cells only showed enrichment in the T2D male GWAS, suggesting a sex-based heterogeneity in the immune regulation of T2D risk.
We also assessed whether sex-specific differentially accessible chromatin regions lie within T2D risk loci.In total, 40 regions that were differentially accessible across sex (FDR < 0.1) overlapped with variants from 37 unique T2D risk signals (Figure 7b).One differentially accessible chromatin region, in particular, was only detected in female lymphocytes, with no detectable reads in male lymphocytes (b38; 19:19627169-1962913019)    (Figure 7c).The differentially accessible female lymphocyte region overlaps with 4 T2D variants at the TM6SF2 risk locus (index variant rs188247550).We found differentially accessible regions in male delta cells to overlap with T2D associated variants in GCK, KCNQ1, PIK3R1, in contrast to females (Figure 7c and Extended Data Fig. 7c).We also found GLI2 to overlap in female ductal cells, in contrast to males (Extended Data Fig. 7d).
Similarly, in the case of male endothelial cells, we found differentially accessible regions to overlap with variants regulating HNF1A, NEUROG3, and in case of acinar cells SLC30A8 (Figure 7c).Previously, 31 variants across 28 T2D risk loci were reported to have sex-specific effects on T2D in a trans-ancestry GWAS, including one variant near TM6SF2 (rs8107974), two variants at GLI2 (rs11688931, rs11688682), and one variant at KCNQ1 (rs2237895). 36Inclusion of two additional T2D meta-analyses which included the X-chromosome found no additional overlap in T2D risk loci with differentially accessible chromatin regions on the X-chromosome. 37,38

Discussion
Our study provides a single cell atlas of sex-specific genomic differences in pancreatic islet cell types in subjects with and without T2D.In non-diabetic islet cells, sex differences in sex-linked genes predominate.In females, XIST and its negative regulator TSIX are upregulated across all islet cells, suggesting a role of X-chromosome dosage compensation 39 in human islet function.Similarly, the Y-linked ubiquitin specific peptidase USP9Y 40 and S4 ribosomal protein RPS4Y1 41 genes are expressed exclusively in all male cells, also suggesting a role for these genes in male islet function.Most genes on one X chromosome of XX cells are silenced in development through X chromosome inactivation by XIST, thus normalizing X chromosome genes dosage between sexes.However, some X chromosome genes escape inactivation and are expressed from both alleles in XX cells. 42,43se "X-escape genes" are conserved between mouse and humans, and several are epigenetic remodelers that promote histone modification to regulate genome access to transcription factors.For example, the histone demethylase KDM6A escapes X inactivation 44 and was more accessible and expressed in female β and α cells.
KDM6A promotes sex differences in T cell biology. 45Similarly, KDM5D is only expressed from the male Y chromosome and was overexpressed in male β and α cells.7][48][49] Thus, sex differences in expression of chromatin remodelers like KDM6A or KDM5D may influence sex-specific chromatin access to transcription factors promoting sex differences in islet function.Consistent with this possibility, we observed a five-to-ten-fold greater number of transcription factorspecific accessible motifs in female compared to male α and β cells.
Non-diabetic female islets exhibited greater ATP-mediated respiration and coupling efficiency than those of males, which is consistent with females' mitochondria having greater functional capacity. 50,51In contrast, female β cells from T2D donors showed reduced activation of pathways enriched in mitochondrial function compared to female β cells from non-diabetic donors, which was not observed in male β cells.In addition, in comparison of T2D vs. non-diabetic β cells, females exhibited seven to ten-times more dysregulated autosomal genes than males.Taken together this suggests that females β cells are resilient and must develop more severe dysfunction to fail than those of males.This is consistent with the observation that female mouse islets retain greater β cell function during metabolic stress. 52Thus, in the transition from normoglycaemia to T2D, female β cell develop greater mitochondrial dysfunction than those of males.This may explain why males are more prone to β cell failure than females as discussed in the introduction.Sex hormones may explain these differences, as estrogen and androgen receptors affect mitochondrial function in female and male β cells. 53,54However, since differences between islets from non-diabetic and T2D donors were present outside of the in vivo hormonal environment, cell autonomous factors, such as the sexually dimorphic sex chromosomes genes described above are more likely to be involved in these differences.
6][57] In addition, non-diabetic black male islets exhibit decreased cAMP-stimulated insulin secretion compared to white male islets.5] In genomic enrichment analyses of our snATAC-seq open chromatin regions for T2D GWAS, we find that differentially accessible regions overlap with T2D-associated variants in a sex-and cell-specific manner.One accessible chromatin region in female lymphocytes overlaps with 4 T2D-associated variants at the TM6SF2 risk locus and was not detectable in male lymphocytes.Previously, 31 variants across 28 T2D risk loci were reported to have sex-specific effects on T2D in a trans-ancestry GWAS, including one variant near the same TM6SF2 locus. 36We also found differentially accessible regions to overlap with classical T2D variants in male but not female δ cells (GCK, KCNQ1 and PIK3R1), endothelial cells (HNF1A and NEUROG3), and acinar cells (SLC30A8).Surprisingly no region overlapped with T2D variants in β cells.
A strength of our study is the use of 'pseudo-bulk' profiles aggregated per cell type in each sample.Collapsing cell profiles by sample enables to effectively control for pseudo-replication due to cells being sampled from a fixed number of donors, whereas treating each cell from the same cluster as an independent observation leads to inflated p-value and spurious results.This approach has demonstrated high concordance with bulk RNA-seq, proteomics and functional gene ontology data. 58,59We applied a hypergeometric statistical model using 'pseudobulk' count data correcting for library composition bias and batch effects in the scRNA-seq. 25This approach has enabled us to recapitulate biological ground truth, where we demonstrate high concordance between accessible chromatin and associated active genes across human islet cells.
In conclusion, this study establishes an integrated accessible chromatin and transcriptional map of human islet cell types across sex and race at single cell resolution, reveals that sex-specific genomic differences in nondiabetic individuals predominantly through sex chromosome genes, and reveals genomic differences in islet cell types in T2D which highlights mitochondrial failure in females.

Limitations of the study
Despite the inclusion of seven black donors (Tulane dataset) to promote genetic diversity, our study is limited by the small sample size.Future extramural funding for the inclusion and study of diverse genetic datasets is essential.Another key consideration is library composition bias owing to targeted islet sequencing, which is not a representation of all pancreatic cells, cell subtypes, or spatiotemporal domains. 60,61Even after utilizing a stringent ambient RNA correction methodology, invariably residual contaminant RNA can be observed across cells.Emphasis is given on generating tools to adjust for ambient RNA particularly in case of pancreatic cells containing high expression of genes such as INS and PRSS1.Differentially accessible chromatin peak counts have FDR adjusted q-value<0.1,GO pathways have FDR adjusted q-value<0.2.measurements for incretin driven insulin secretion measurements outlined in (g).i, Oxygen consumption ratio for islets across sex and race.j, Basal respiration, glucose mediated respiration, maximal (max) respiration, ATP mediated respiration, non-electron transport chain (ETC) respiration and coupling efficiency, across sex and race.k, Oxygen consumption ratio for islets across sex.l, Basal respiration, glucose mediated respiration, maximal (max) respiration, ATP mediated respiration, non-electron transport chain (ETC) respiration and coupling efficiency, of human islets across sex.*pval < 0.05, **pval < 0.01 is significant.n = 15 (non-diabetic).

Figures
Figures

Figure 1 :
Figure 1: Pancreatic islet cells have a conserved expression signature across sex and race.

Figure 2 :
Figure 2: Transcriptional differences across islet β and α cells, highlight enrichment in sex-chromosome genes.a, Principal component analysis (PCA) plot of pseudo-bulk transcriptional profiles across all individual donor islets.b, PCA plot of pseudo-bulk transcriptional profiles in each cell type across all donors.c-d, Volcano plots showing all differentially expressed genes (DEGs) (left panel) or autosomal DEG subset (right panel) across sex in non-diabetic: c, β cells.d, α cells.e, GO analysis of all β cell DEGs.f, GO analysis of all α cell DEGs.n= 36 non-diabetic and n=16 T2D diabetic donors.DEGs have FDR adjusted q-value <0.1, GO pathways have FDR adjusted q-value <0.2

Figure 3 :
Figure 3: Chromatin accessibility landscape of human pancreatic islet cell types.a, UMAP plot denoting integrated clustering of 52,613 single pancreatic islet cells across 11 clustered cell types based on their accessible chromatin profiles, spanning n=15 datasets.Each cluster cell type is denoted by a label and color.b, Cell diversified based on sex and race.c, Cell distribution stemming from each of the n=15 donors, grouped based on race and sex.d, Normalized confusion matrix, showing correlation across cell types

Figure 4 .
Figure 4. Sex-based enrichment for sex-chromosome gene accessibility in human islet cells 449

Figure 5 .
Figure 5. Sex and race differences in islet hormone secretion and bioenergetics.a, Dynamic insulin secretion assay, showing response to 16.7mM glucose, IBMX + 16.7mM Glucose, epinephrine + 1.7mM Glucose and potassium chloride + 5.6mM glucose.Each curve represents secretion normalized to total insulin content across sex and race.b, Area under the curve (AUC) measurements for incretin driven insulin secretion measurements outlined in (a).c, Dynamic insulin secretion assay, showing response to 16.7mM glucose, IBMX + 16.7mM Glucose, epinephrine + 1.7mM Glucose and potassium chloride + 5.6mM glucose.Each curve represents secretion normalized to total insulin content across sex.d, Area under the curve (AUC) measurements for incretin driven insulin secretion measurements outlined in (b).e, Dynamic glucagon secretion assay, showing response to 16.7mM glucose, IBMX + 16.7mM Glucose, epinephrine + 1.7mM Glucose and potassium chloride + 5.6mM glucose.Each curve represents secretion normalized to total glucagon content across sex and race.f, Area under the curve (AUC) measurements for incretin driven insulin secretion measurements outlined in (e).g, Dynamic glucagon secretion assay, showing response to 16.7mM glucose, IBMX + 16.7mM Glucose, epinephrine + 1.7mM Glucose and potassium chloride + 5.6mM glucose.Each curve

Figure 6 .
Figure 6.Transcriptional differences in T2D compared to non-diabetic endocrine cells.a, Heatmap of DEGs across T2D donors.b, Violin plots showing DEGs across male and female T2D β/α cells.c, Violin plots showing DEGs across β/α cells when diabetic donors are compared to non-diabetic controls across sex.d, Venn diagram showing DEGs across different sex-disease comparisons in case of β cells.Color denotes the number of genes.e, Gene ontology dotplot for upregulated and downregulated pathways for β-cell DEGs.f, Venn diagram showing DEGs across different sex-disease comparisons in case of α cells.Color denotes the number of genes.g, Gene ontology dotplot for upregulated and downregulated pathways for α-cell DEGs.n= 36 non-diabetic and n=16 T2D diabetic donors.DEGs have FDR adjusted q-value<0.01,GO pathways have FDR adjusted q-value<0.2

Figure 7 .
Figure 7. GWAS utilizing the DIAMANTE Type 2 diabetes dataset shows cell type and sex specific variants influencing Type 2 Diabetes risk